Isocytosine deaminase Vcz as a novel tool for the prodrug cancer therapy

Abstract Background The cytosine deaminase (CD)/5-fluorocytosine (5-FC) system is among the best explored enzyme/prodrug systems in the field of the suicide gene therapy. Recently, by the screening of the environmental metagenomic libraries we identified a novel isocytosine deaminase (ICD), termed Vcz, which is able of specifically converting a prodrug 5-fluoroisocytosine (5-FIC) into toxic drug 5-fluorouracil (5-FU). The aim of this study is to test the applicability of the ICD Vcz / 5-FIC pair as a potential suicide gene therapy tool. Methods Vcz-expressing human glioblastoma U87 and epithelial colorectal adenocarcinoma Caco-2 cells were treated with 5-FIC, and the Vcz-mediated cytotoxicity was evaluated by performing an MTT assay. In order to examine anti-tumor effects of the Vcz/5-FIC system in vivo, murine bone marrow-derived mesenchymal stem cells (MSC) were transduced with the Vcz-coding lentivirus and co-injected with 5-FIC or control reagents into subcutaneous GL261 tumors evoked in C57/BL6 mice. Results 5-FIC alone showed no significant toxic effects on U87 and Caco-2 cells at 100 μM concentration, whereas the number of cells of both cell lines that express Vcz cytosine deaminase gene decreased by approximately 60% in the presence of 5-FIC. The cytotoxic effects on cells were also induced by media collected from Vcz-expressing cells pre-treated with 5-FIC. The co-injection of the Vcz-transduced mesenchymal stem cells and 5-FIC have been shown to augment tumor necrosis and increase longevity of tumorized mice by 50% in comparison with control group animals. Conclusions We have confirmed that the novel ICD Vcz together with the non-toxic prodrug 5-FIC has a potential of being a new enzyme/prodrug system for suicide gene therapy

Inflammation Control and Immunotherapeutic Strategies in Comprehensive Cancer Treatment

Tumor growth and expansion are determined by the immunological tumor microenvironment (TME). Typically, early tumorigenic stages are characterized by the immune system not responding or weakly responding to the tumor. However, subsequent tumorigenic stages witness the tumor promoting its growth and metastasis by stimulating tumor-protective (pro-tumor) inflammation to suppress anti-tumor immune responses. Here, we propose the pivotal role of inflammation control in a successful anti-cancer immunotherapy strategy, implying that available and novel immunotherapeutic modalities such as inflammation modulation, antibody (Ab)-based immunostimulation, drug-mediated immunomodulation, cancer vaccination as well as adoptive cell immunotherapy and donor leucocyte transfusion could be applied in cancer patients in a synergistic manner to amplify each other’s clinical effects and achieve robust anti-tumor immune reactivity. In addition, the anti-tumor effects of immunotherapy could be enhanced by thermal and/or oxygen therapy. Herein, combined immune-based therapy could prove to be beneficial for patients with advanced cancers, as aiming to provide long-term tumor cell/mass dormancy by restraining compensatory proliferation of surviving cancer cells observed after traditional anti-cancer interventions such as surgery, radiotherapy, and metronomic (low-dose) chemotherapy. We propose the Inflammatory Prognostic Score based on the blood levels of C-reactive protein and lactate dehydrogenase as well as the neutrophil-to-lymphocyte ratio to effectively monitor the effectiveness of comprehensive anti-cancer treatment

1,4-dihidropiridino darinių poveikis žiurkės pėdos edemai, gliukokortikoidinių hormonų sintezei ir jų sąveika su gliukokortikoidų receptoriais aktyvumui

The influence of the 1,4-dihydropyridine derivatives OSI-7725 and OSI-7727 on acute inflammation, glucocorticoid hormone (OH) synthesis and specific GH binding to glucocorticoid receptor (GR) activity was investigated. Since several 1,4-dihydropyridines (DHPs) possess anti-inflammatory properties, we first studied the effect of racemate OSI-7725 and its (+) enantiomer OSI-7727 in a model of rat paw edema induced by carrageenan. These compounds had a preventative effect in this model of inflammation and decreased edema development at a most effective dose of 0.1 mg/kg by 26.6% and 41.6%, respectively. Both compounds were ineffective when added after carrageenan injection. Subsequent experiments in vivo showed that OSI-7725 and OSI-7727 at a dose of 5 mg/kg stimulated GH synthesis in rat adrenal. They induced an increase in total, transcortin-bound and free corticosterone levels in plasma 2 h following intraperitoneal administration. The pretreatment with metyrapone, an inhibitor of steroid synthesis, abolished the stimulatory effects of the compounds tested, indicating a steroidai component in the mechanism of action of DHPs. Further experiments in vitro showed that these DHPs inhibited dexamethasone (DEX) binding to GR. More active in this model was OSI-7725. It inhibited specific ligand-receptor binding by 80% at concentrations 10-5 and 10-4 M. OSI-7727 was active only at a concentration of 10-4 M. 1C50 values of these compounds were 3.59 and 5.52 μM, respectively. The results show a significance of these DHPs in the regulation of vitally important processes and provide an evidence of involvement of fine mechanisms through which the pharmacological effects of DHPs can be released. This could extend the potential of the application of these substances

Stromal vascular fraction cells for the treatment of critical limb ischemia: a pilot study

Abstract Background Cell-based therapy is being explored as an alternative treatment option for critical limb ischemia (CLI), a disease associated with high amputation and mortality rates and poor quality of life. However, therapeutic potential of uncultured adipose-derived stromal vascular fraction (SVF) cells has not been evaluated as a possible treatment. In this pilot study, we investigated the efficacy of multiple injections of autologous uncultured adipose-derived SVF cells to treat patients with CLI. Methods This study included 15 patients, from 35 to 77 years old, with rest pain and ulceration. SVF cells were injected once or twice in the ischemic limb along the arteries. Digital subtraction angiography was performed before and after cell therapy. The clinical follow up was carried out for the subsequent 12 months after the beginning of the treatment. Results Multiple intramuscular SVF cell injections caused no complications during the follow-up period. Clinical improvement occurred in 86.7% of patients. Two patients required major amputation, and the amputation sites healed completely. The rest of patients achieved a complete ulcer healing, pain relief, improved ankle-brachial pressure index and claudication walking distance, and had ameliorated their quality of life. Digital subtraction angiography performed before and after SVF cell therapy showed formation of numerous vascular collateral networks across affected arteries. Conclusion Results of this pilot study demonstrate that the multiple intramuscular SVF cell injections stimulate regeneration of injured tissue and are effective alternative to achieve therapeutic angiogenesis in CLI patients who are not eligible for conventional treatment. Trial registration number at ISRCTN registry, ISRCTN13001382. Retrospectively registered at 26/04/2017

Application of stem cells and synthetic scaffolds for tissue engineering

Biochemijos katedraKauno medicinos universitetasKauno medicinos universiteto Kardiologijos institutasKauno medicinos universiteto Kardiologijos institutas, [email protected] Didžiojo universiteta

Scaffold design for artificial tissue with bone marrow stem cells

Objective: The aim of this study was to test polymeric materials (collagen, fibrin, polyimide film, and polylactic acid) for single- and multi-layer scaffold formation. Materials and methods: In our study, we used rabbit bone marrow stem cells (rBMSCs) and human mesenchymal stem cells (hMSCs) with materials of a different origin for the formation of an artificial scaffold, such as a collagen scaffold, fibrin scaffold produced from clotted rabbit plasma, electrospun poly(lactic acid) (PLA) mats, polyimide film (PI), and the combination of the latter two. Cell imaging was performed 3–14 days after cell cultivation in the scaffolds. Time-lapse imaging was used to determine hMSC mobility on the PI film. Results: Cell incorporation in collagen and clotted fibrin scaffolds was evaluated after 2-week cultivation in vitro. Histological analysis showed that cells penetrated only external layers of the collagen scaffold, while the fibrin clot was populated with rBMSCs through the entire scaffold thickness. As well, cell behavior on the laser micro-structured PI film was analyzed. The mobility of hMSCs on the smooth PI film and the micro-machined surface was 20 ± 2 μmm/h and 18 ± 4 μmm/h, respectively. After 3-day cultivation, hMSCs were capable of spreading through the whole 100 ± 10 μmm-thick layer of the electrospun PLA scaffold and demonstrated that the multilayer scaffold composed of PI and PLA materials ensured a suitable environment for cell growth. Conclusions: The obtained results suggest that electrospinning technology and femtosecond laser micro-structuring could be employed for the development of multi-layer scaffolds. Different biopolymers, such as PLA, fibrin, and collagen, could be used as appropriate environments for cell inhabitation and as an inner layer of the multi-layer scaffold. PI could be suitable as a barrier blocking cell migration from the scaffold. However, additional studies are needed to determine optimal parameters of inner and outer scaffold layers

Egzosomų adhezija naudojant funkcionalizuotus nanodeimantus

Edukologijos tyrimų institutasMokytojų rengimo institutasNacionalinis vėžio institutas, VilniusVytauto Didžiojo universiteta